This invention relates to new DNA sequences, a method for producing new plants which contain a new DNA sequence, the coding sequence thereof being expressed after ozone induction. The invention also relates to said new plants and the use of DNA sequences to produce ozone-responsive gene expression in plants and plant cells. Moreover, it relates to a new promotor, the specificity of which is increased by removal of the ozone response capacity thereof.
The ozone concentrations in the lower troposphere above the continents of the northern hemisphere have steadily increased over the past one hundred years as a result of greater industrial activities (Volz and Kley (1988) Nature 332, 240-242). Meanwhile, ozone values reach intermittent peak concentrations of 100 nL/L up to nL/L in Europe and North America (Krupa et al. (1995) Environ. Pollut: 87, 119-126).
The phytotoxicity of the air pollutant ozone has been well tested and documented, e.g., in Heagle (1989) Annu. Rev. Phytopathol. 27, 397-423; Heath (1994) in: Alscher, Wellburn (ed) xe2x80x9cPlant responses to the gaseous environmentxe2x80x9d, pp. 121-145, Chapman and Hall, London. A decreased net photosynthesis and an increased early senescence are usually the result of such ozone impact which, consequently, results in diminished plant growth and a lower harvest yield.
Although ozone penetrates the plant cell through open stomata by means of diffusion, the ozone concentration in the intercellular spaces of the leaf is almost zero, irrespective of the environmental ozone concentration (Laisk et al. (1989) Plant Physiol. 90, 1163-1167). It is currently assumed that ozone reacts quickly with components of the cell walls and the plasmalemma and is converted into reactive oxygen species, such as peroxide-anions, hydroxyl radicals and hydrogen peroxide which were detected in ozone-treated plant material by using electron spin resonance spectroscopy (Mehlhorn et al. (1990) Physiologia Plantarum 79, 377-383). The so-called xe2x80x9coxidative burstxe2x80x9d, i.e. the fast development of a relatively high quantity of reactive oxygen species, can lead to a dramatic disturbance of the normal cell function due to alteration of the permeability of the plant membrane, inactivation of redox-sensitive proteins and increased lipid peroxidation.
Recent tests conducted on ozone-treated plants showed an increased biosynthesis of non-specific, defensive enzymes, the function of which is to protect live cells against damage due to oxidative stress (Kangasjxc3xa4rvi et al. (1994 Plant, Cell and Environment 17, 783-794). Yet the signal-transduction chain, which is responsible for the ozone-induced gene activation, which transmits to the cell core the relevant information about the formation of apoplastic, reactive oxygen species, has not been understood up to now. Various factors, such as the increase of calcium concentration in cytosol (Price et al. (1994) The Plant Cell 6, 1301-1310), the formation of salicylic acid (Klessig and Malamy (1994) Plant Mol. Biol. 26, 1439-1458) and the phytohormone jasmon acid (Farmer (1994) Plant Mol. Bio. 26 1423-1437) and ethylene (Ecker (1995) Science 268, 667-674) are currently being discussed as possible signal connections, caused by oxidative stress, which generally play a part in defence reactions of plants.
Tests conducted on ozone-gassed tobacco plants showed that ozone causes an increased expression of various disease-resistant genes, namely a few PR-(pathogenesis-related) proteins (Ernst et al. (1992) Plant Mol. Biol. 20, 673-682; Ernst et al. (1996) J. Plant Physiol. 148, 215-221; Eckey-Kaltenbach et al. (1994) Plant Physiol. 104, 67-74). These results indicate that oxidative stress, caused by ozone, influences the expression and regulation of defensive genes of plants in a similar way as that described regarding pathogenic attack. Only very limited information on cis-regulatory elements and transcription factors, which possibly play a part in the control of gene expression of non-specific defensive genes as a response to various environmental influences, is available at present (Lee et al. (1994) Eur. J. Biochem. 226, 109-114). However, based on previous results, it can be assumed that with respect to the genes coding for PR-proteins, separate or at least only partly overlapping ways of signal transduction exist (Somssich (1994) in: Nover (ed) xe2x80x9cPlant promoters and transcription factorsxe2x80x9d, pp. 163-179, Springer Publishing House, Berlin; Dolferus et al. (1994) Plant Physiol. 105, 1075-1087).
With respect to the activity of the stilbene synthase (STS), which takes part in the phytoalexin synthesis, it is known that in adult plants it is induced by environmental stress factors, such as, e.g., pathogenic attack (Langcake (1981) Physiol. Plant Pathol. 18, 213-226), ultraviolet light (Fritzemeier and Kindl (1981) Planta 151, 48-52) and ozone (Rosemann et al. (1991) Plant Physiol. 97, 1280-1286. Contrary to this a constitutive expression pattern was observed in embryos (Sparvoli et al. (1994) Plant Mol. Biol. 24, 743-755).
Stilbene synthase enzymes catalyze the synthesis of stilbenes such as resveratrol or pinosylvin from one molecule of p-cumaroyl-CoA or cinnamoyl-CoA and three units of malonyl-CoA. Resveratrol as well as pinosylvin have photoalexin properties and an antifungal activity, and perform, as phytoalexins in combination with other stilbenes derived from the phenylpropane metabolism, an important function in the defence against pathogens (Hart (1981) Annu. Rev. Phytopathol. 19, 437-458).
STS genes are found in some non-related plant species such as, e.g., peanut (Schrxc3x6der et al. (1988) Eur. J. Biochem. 172, 161-169), grapevine (Hain et al. (1993) Nature 361, 153-156) and pine (Fliegmann et al. (1992) Plant Mol. Biol. 18, 489-503) and are organized in larger gene families, comprising six or more genes (Lanz et al. (1990) Planta 181, 169-175; Wiese et al. (1994) Plant Mol. Biol. 26, 667-677).
Experiments with transgenic tobacco cells indicate that the expression of the stilbene synthase is regulated mainly at a transcription level, and that the stress-induced signal transduction chain has been preserved in various plant species during the course of evolution (Hain et al. (1990) Plant Mol. Biol.15, 325-335.
STS genes from peanut (Arachis hypogaea) and grapevine (Vitis vinifera) have already been isolated (Schrxc3x6der et al. (1988) supra or Hain et al. (1993) supra) and expressed in transgenic plants (Hain et al. (1990) supra or Hain et al. (1993) supra).
DNA sequences coding for stilbene synthase are known, e.g., from European Patent EP 0 309 862, German Patent Application DE-A-41 07 396, European Patent Application 0 464 461, as well as U.S. Pat. No. 5,500,367. These documents describe the isolation of stilbene-synthase genes and their use to produce transgenic plants. The resulting transgenic plants show greater resistance to various plant pests such as fungi, bacteria, insects, viruses and nematodes. Plasmids containing STS genes have been deposited with the German Collection of Microorganisms (DSM), Mascheroder Weg 1B, D-38124 Braunschweig. Also included in the deposition is the VstI-gene from grapevine in the pVstI plasmid, under deposit number DSM 6002 (DE-A-41 07 396, EP-A-0 464 461, U.S. Pat. No. 5,500,367).
While in the meantime also the use of STS coding sequences to produce male, sterile plants and altered blossom colour has been described (German Patent Application DE-A-44 40 200), a possible relation between STS gene expression and ozone induction has remained completely unexplored up to now.
Meanwhile there are various indications that in order to produce an effective as possible resistance to disease, based on the expression of STS genes in transgenic plants, it is advantageous if the expression of the heterologous STS gene (or the heterologous STS genes) in the plant is stimulated first of all by the attacking pathogen, i.e. if it is stimulated first of all by the interaction of plant and pathogen (Fischer and Hain (1994) Current Opinion in Biotechnology 5, 125-130; Fischer (1994) xe2x80x9cOptimization of the heterological Expression of Stilbene-synthase Genes for the Protection of Plantsxe2x80x9d, Hohenheim University). This is endorsed particularly by the observation that the pathogen-induced STS gene expression is locally limited to the place of infection and is of a transient nature, which means that the STS expression rises relatively fast to a maximum and declines again in less than 48 hours (Hain et al. (1993) supra). Also experiments conducted on transgenic tobacco plants, in which STS genes were expressed under the constitutive 35S RNA promotor of cauliflower mosaic virus, showed that the resistance to disease achieved in said plants is lower than in plants which expressed the identical genes under the control of the pathogen-inducible homologous STS promotor after fungus infection (Fischer and Hain (1994) supra; Fischer (1994) supra). Anyway, it is desirable that the STS expression in transgenic, cultivated plants which, due to the inserted STS genes show greater resistance to disease, are activated and controlled solely (and not until) by the attack of pathogens and not additionally (or already before) by undesirable environmental stress factors such as ozone.
Thus, it is an important task of biotechnological research of plant protection to realize a more specific expression of defensive genes in plants, in order to be able to materialize molecular biological strategies for producing plants of greater resistance in an efficient and controllable manner. An important aspect in doing so is to eliminate undesirable, non-specific environmental stimuli such as, e.g., the induction of certain defensive genes through ozone, ultraviolet light, heavy metals, extreme temperatures and other abiotic stimuli.
Thus, it is an important object of this invention to make available new DNA sequences which play a direct part in the ozone-induced expression of resistance genes.
Another object of the invention is to show possibilities for removing the ozone induction, i.e. to eliminate undesirable stimulation of the gene expression through ozone.
Furthermore, an important object of this invention is to provide a DNA sequence with the help of which stilbene-synthase genes can be expressed in transgenic plants only after contact of the plant with the pathogen and not through ozone stimulation.
As mentioned already at the outset, a steady increase in ozone impact can be observed which also has drastic effects on vegetation. The observation and determination of ozone concentrations in the air constitute already today a focal point of chemical, physical and biological, environmental research. An important instrument in this context are the so-called biomonitors, with the help of which ozone impact and the consequences thereof, particularly phytotoxic effects can be easily determined both qualitatively and quantitatively.
Thus, a further object of this invention is to provide DNA sequences that can be used to produce targeted ozone-inducible promoters. With the help of such promotors it is possible for so-called reporter genes, the expression of which can be proven by simple, enzymatic tests, and which are well known in biotechnical research, to be used as biomonitors.
Another object of the invention is to provide a system with the help of which certain genes,xe2x80x94whose gene products are able to detoxify oxygen species in cellsxe2x80x94can be xe2x80x9cturned onxe2x80x9d, if necessary, as for instance in case of great ozone impact. In other words, by providing DNA sequences, which are responsible for ozone-responsive gene regulation, an ozone-inducible expression of said genes such as, e.g., catalase and/or superoxide-dismutase genes, shall be rendered possible. Thus, it is an object of the invention to make available DNA sequences which can be used for producing an ozone-inducible, cellular xe2x80x9cozone protection systemxe2x80x9d. Further objects of the invention will become apparent as the following description proceeds.
These problems are solved by the subject-matter of the independent claims, based particularly on the provision of the DNA sequences, according to the invention, which are directly involved in the ozone-induced gene expression in plants.
We found, to our surprise, that a certain plant nucleic acid sequence is directly involved in the ozone-responsive STS expression. While experiments with transgenic tobacco embryos and plantsxe2x80x94which express the customary reporter gene uidA from E. coli that codes for a xcex2-glucuronidase under the control of variously long 5xe2x80x2 deletions of the VstI-promotor from grapevinexe2x80x94indicate that at least a few cis-elements, responsible for the fungus induction, are within the range of the promotor which comprises base pairs xe2x88x92140 to xe2x88x92280 (calculated from the start of transcription) (Fischer (1994) supra), the range of the VstI sequence, which comprises base pairs xe2x88x92280 to xe2x88x92430, is essential for a strong activation of the gene expression through ozone. Based on our experiments, it was possible to show that a VstI promoter, which is left with only base pairs up to and including xe2x88x92280 (and which thus is lacking the VstI-promoter sequences, located further upstream) is no longer ozone inducible. As mentioned above, said shortened promotor is nevertheless still able to indicate pathogen-induced gene expression of the coding sequence controlled by same (see Fischer (1994) supra).
Our analyses also lead us to suspect a relation between the treatment of plants with ozone and an increased biosynthesis or release of ethylene in plant cells. Therefore, an involvement of ethylene-responsive elements in the ozone-induced gene expression cannot be excluded. Thus, by taking into consideration familiar cis-elements, which are currently being discussed in connection with ethylene-response capacity (Sessa et al. (1995) Plant Mol. Biol. 28, 145-153; Shinshi et al. (1995) Plant Mol. 27, 923-932), an involvement of the sequence range of the VstI promotor, which comprises base pairs xe2x88x92283 to xe2x88x92273, cannot be excluded in an ozone-induced STS-gene expression.
Accordingly, the ozone-responsive DNA sequence range, which is described here for the first time, comprises base pairs xe2x88x92270 to xe2x88x92430 of the VstI promotor from grapevine.
Thus, the present invention relates to the DNA sequence (SEQ ID NO:1), as defined in Claim 1:
ACTTTTCGAG CCCCTTGAAC TGGAAATTAA TACATTTTCC ACTTGACTTT TGAAAAGGAG GCAATCCCAC GGGAGGGAAG CTGCTACCAA CCTTCGTAAT GTTAATGAAA TCAAAGTCAC TCAATGTCCG AATTTCAAAC CTCANCAACC CAATAGCCAA T, which is essential for the ozone-induced gene expression in plants. A preferred version of the DNA sequence, according to the invention, deals with a DNA sequence, which originates from grapevine, and especially preferred from the stilbene synthase gene VstI from grapevine (base pairs xe2x88x92270 to xe2x88x92430).
Furthermore, the invention relates to a promotor region of the VstI-gene which lacks at least the DNA sequence that comprises base pairs xe2x88x92270 to xe2x88x92430 of the VstI-gene. A preferred version concerns a promotor region of the VstI gene, which only comprises the sequence range from the start of the translation to base pair xe2x88x92270 of the VstI gene. It is particularly preferred that the promoter region, which lacks the sequence range xe2x88x92270 to xe2x88x92430 of the VstI gene, is able to convey a pathogen-induced gene expression in plants.
The invention also relates to chimeric nucleic acid molecules, into which the DNA sequence of base pairs xe2x88x92270 to xe2x88x92430 of the VstI-gene or at least a fragment of this sequence range has been inserted. It is especially preferred that the chimeric nucleic acid molecules, according to the invention, render possible, due to the presence of the DNA sequence of base pairs xe2x88x92270 to xe2x88x92430 of the VstI gene or at least a fragment thereof, an ozone-inducible expression of the coding regions in plants contained therein.
The nucleic acid molecules can be any nucleic acid molecules, especially DNA or RNA molecules, e.g., cDNA, genomic DNA, mRNA, etc. They can be naturally occurring molecules or molecules produced by gene technology or by chemical synthetic processes.
By making available, according to the invention, DNA sequences, promotor regions, nucleic acid molecules or vectors, it is now possible to mutate plant cells by means of gene technology methods in such a way that they show ozone-inducible characteristics. Furthermore, it is now possible to mutate plant cells by means of gene technology methods in such a way that they characterize one or more genesxe2x80x94which are naturally ozone inducible, due to the presence of the DNA sequence set out in Claim No. 1 or a DNA sequence derivable therefrom or one that is homologous with said DNA sequencexe2x80x94as being no longer inducible by ozone but preferably inducible mainly by pathogens.
A special advantage of the invention is the fact that the ozone induction of naturally ozone-inducible genes in plants and plant cells can be eliminated by deleting the DNA sequence, as set out in Claim No. 1, or at least a fragment thereof, in the genes which naturally contain this DNA sequence or a DNA sequence which can be derived therefrom or one that is homologous with said DNA sequence.
Another advantage of the invention is that genes which cannot or cannot substantially be naturally induced through ozone, can be characterized as being ozone inducible by using the invented nucleic acid sequences in plant and plant cells. In a preferred version the nucleic acid sequence, which is responsible for the ozone-inducible expression or at least a fragment thereof, controls the expression of genes, the gene products of which are able to detoxify reactive oxygen species that can develop among other things, as a consequence of ozone in plant cells. In a particularly preferred version, the nucleic acid sequence controls the expression of catalase and/or superoxide-dismutase genes.
In an alternative version, the DNA sequence that is responsible for the ozone-inducible gene expression controls the expression of reporter genes which are measured in order to determine ozone concentrations quantitatively and/or qualitatively and to evaluate the effects of ozone. Such reporter genes can be, e.g., the uidA gene from E. coli, which codes for the enzyme xcex2-glucuronidase (GUS), luciferase genes or other genes, customary in plant biotechnology. Every expert in biotechnology, biochemistry or molecular biology is familiar with appropriate reporter genes.
Furthermore, the invention relates to vectors which contain the above-mentioned DNA sequences or promoter regions or fragments thereof. Thus, this invention relates also to vectors, particularly plasmids, cosmids, viruses, bacteriophages and other vectors, common in gene technology, which contain the above-mentioned nucleic acid molecules, according to the invention and which, if required, can be used for transferring said nucleic acid molecules to plants or plant cells.
The invention also relates to transformed microorganisms, such as bacteria, viruses and fungi which contain the nucleic acid sequences, according to the invention.
It is also an object of the invention to provide plants and plant cells which are characterized by the absence of the ozone-inducible expression of genes that are naturally induced in plants through ozone.
This problem is solved by providing the DNA sequence responsible for the ozone induction and by making available promoters which lack said sequence and which for this reason render possible a no longer ozone-inducible gene expression of genes in plant and plant cells controlled by the promotors.
The problem of rendering possible an ozone-responsive gene expression of genes which are not naturally inducible through ozone is likewise solved by providing the DNA sequence, according to the invention. Thus, plants and plant cells are made available which in the presence of ozone specifically define certain characteristics.
A preferred version concerns transformed plants and plant cells in which genes are ozone-inducibly expressed, and the gene products thereof are able to detoxify reactive oxygen species in plant cells. Particular preference is given in this connection to catalase and/or superoxide-dismutase genes. As an alternative, it is possible to produce plants and plant cells (including protoplasts) which characterize so-called reporter genes after induction through ozone and which, if required, can be used as biomonitors.
Thus, the subject-matter of the invention is transgenic plants which contain, integrated in the plant genome, the recombined nucleic acid molecules, as described above. Such plants can, in principle, be any plants. It concerns preferably a monocotyle or dicotyle useful plant. Examples of monocotyle plants are plants which belong to the genuses of Avena (oat), Triticum (wheat), Secale (rye), Hordeum (barley), Oryza (rice), Panicum, Pennisetum, Setaria, Sorghum (millet), Zea (corn). Dicotyle, useful plants are, e.g., cotton, leguminous plants such as legumes, and especially alfalfa, soya bean, rape, tomato, sugar beet, potato, ornamental plants, trees. Other useful plants can be fruit-bearing plants (particularly apples, pears, cherries, grapes, citrus fruits, pineapples and bananas), oil palms, tea and cacao shrubs, tobacco, sisal, as well as medicinal plants, such as rauwolfia and digitalis. Special preference is given to grain, such as wheat, rye, oat, barley, rice, corn and millet, sugar beet, rape, soya, tomato, potato and tobacco.
Furthermore, the subject-matter of the invention is the propagation material of plants, according to the invention, such as, e.g., seeds, fruits, cuttings, tubers, rootstock, etc., as well as constituents of such plants, such as plant cells, protoplasts and calli.
The plant cells include differentiated and non-differentiated plant cells (including protoplasts), as well as plant cells (including protoplasts) in which nucleic acid molecules are integrated in the plant genome or are present as autonomous molecules (including transient transformation).
In another version, the invention relates to host cells, particularly prokaryontic or eukaryontic cells, which have been transformed or inoculated with a recombined nucleic acid molecule or a vector, as described above, and cells which originate from said host cells and which contain the described nucleic acid molecules or vectors. The host cells can be bacteria or fungus cells, as well as plant or animal cells.
The object of this invention is also to show methods for the production of plant and plant cells which are characterized by the lack of an ozone-inducible expression of a gene, the expression thereof in plants and plant cells is naturally stimulated by ozone.
This problem is solved through processes by means of which the production of new plants and plant cells, which do not have this naturally occurring ozone-induced gene expression, is made possible.
As already mentioned above, it is also the object of this invention to provide methods for the production of plants and plant cells which express such genesxe2x80x94the expression of which is naturally not, or not substantially activated by ozonexe2x80x94after ozone stimulation. This problem is solved by methods with the help of which plants and plant cells can be produced which, after the invented DNA sequences or at least a fragment thereof has been inserted into naturally not ozone-inducible genes, or genes which are ozone-inducible only to a minor degree express such genes after ozone stimulation.
There are various methods by which such new plants or plant cells can be produced. For one thing, plants or plant cells can be mutated by conventional gene-technological transformation methods in such a way that the new nucleic acid molecules are being integrated in the plant genome, which means that stable transformants are produced.
According to the invention, plants or plant cells which, due to the absence of the invented nucleic acid sequence or at least a fragment thereof, no longer show an ozone induction of the gene(s) which naturally contains/contain said sequence, are produced by a method which includes the following steps:
a) Deletion of the DNA sequencexe2x80x94as defined in Claim No. 1, or a sequence which can be derived from said sequence, or which is homologous with said sequence, or at least a fragment of such sequencexe2x80x94from a gene which after the deletion of the invented DNA sequence, includes regulatory elements essential for the possibly regulated transcription and translation in plant cells, and has at least one coding sequence, as well as possibly a termination signal for the termination of the transcription and the addition of a poly-A-tail to the respective transcript.
b) Transformation of plant cells with the gene or nucleic acid molecule, produced in step a), and
c) possibly the regeneration of transgenic plants and possibly the propagation of the plants.
As an alternative it is possible in step a) that instead of deleting the sequence responsible for the ozone-induction, said sequence or at least a fragment thereof can be inactivated or blocked, e.g., through mutagenesis, and thus remain in the gene in an inactivated form. Irrespective of the manner in which the ozone-responsive gene range is deleted, all manipulative measures can be carried out by means of conventional methods and aids of recombined gene technology (see, e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, N.Y.).
In a particularly preferred version the nucleic acid molecule, which in step b) is transferred to plants or plant cells, contains regulatory elements which allow, e.g., a pathogen-induced gene expression of the coding sequence.
According to the invention, plants or plant cells which, due to the presence of the invented nucleic acid sequence that is essential or co-responsible for an ozone-induced expression of the genes containing same, or at least a fragment of said sequence, are produced by a method which includes the following steps:
a). Insertion of at least one DNA sequence, according to the invention, which in plants can produce an ozone-induced gene expression, or a sequence which is derivable from said sequence, or which is homologous with said sequence, or at least a fragment of said sequence, in a gene which is not naturally or not substantially expressed as ozone-inducible.
b) Transformation of plant cells by way of the gene or nucleic acid molecule, produced in step a), which has all elements that are naturally required for the expression in plant cells, and
c) possibly the regeneration of transgenic plants and perhaps the propagation of plants.
In a preferred version the gene concerned is a catalase dismutase, superoxide-dismutase or a common reporter gene.
Another object of the invention is to show advantageous usage of the invented nucleic acid sequences.
The invention includes, therefore, uses of the new DNA molecules to produce the aforementioned plants and plant cells, according to the invention, which are characterized either by the absence of a certain phenotypical distinguishing mark that is normally influenced by ozone, or which precisely due to the presence of the invented DNA sequence distinguish themselves from non-transgenic plants and plant cells by ozone-induced characteristics.
Furthermore, the invention includes the use of the invented nucleic acid molecules to produce plants which are characterized by an increased pathogen-induced but not an ozone-induced resistance to disease.
The invention also relates to the use of the invented nucleic acid sequences or fragments thereof for detecting and identifying ozone-responsive nucleic acid elements.
The expert can identify such ozone-responsive nucleic acid elements by applying customary molecular biological methods, e.g., hybridizing experiments or DNA protein-binding studies. For example, as a first step, the poly (A)+ RNA is isolated from a tissue which was treated with ozone. Then a cDNA-bank is set up. In a second step and with the help of cDNA-clones, which are based on poly(A)+ RNA molecules from a non-treated tissue, those clones from the first bank are identified by way of hybridizing whose corresponding poly(A)+ RNA molecules are induced strictly in the ozone treatment. With the help of cDNAs identified in this manner, promoters which have ozone-responsive elements are subsequently isolated. The nucleic acid sequences and molecules can be useful instruments when examining and characterizing these isolated promoters.
The subject-matter of the invention includes also nucleic acid molecules or fragments thereof which hybridize with one of the above-described nucleic acid molecules or one of the above-mentioned DNA sequences of the invention. Within the scope of this invention the term xe2x80x9chybridizingxe2x80x9d means hybridizing under conventional hybridizing conditions, preferably under stringent conditions, such as are described, for example, in Sambrook et al (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Laboratory Press, Cold Spring Harbor, N.Y.
Nucleic acid molecules which hybridize with the molecules, according to the invention, can be isolated, e.g., from genomic or cDNA banks.
The identification and isolation of such nucleic acid molecules can be accomplished by using the nucleic acid molecules, according to the invention, or fragments of said molecules or the reverse complements of such molecules, e.g., by way of hybridizing according to standard procedure (see, e.g., Sambrook et al, supra).
Thus, the invention relates also to the use of an invented DNA sequence or fragments thereof to identify and isolate homologous sequences from plants or other organisms.
For example, nucleic acid molecules which have exactly or substantially the invented nucleotide sequences or fragments of such sequences can be used as a hybridizing probe. The fragments used as a hybridizing probe can also be synthetic fragments which were produced with the help of customary synthesis techniques and the sequence of which basically corresponds with that of a nucleic acid molecule, according to the invention. Once genes that hybridize with the invented nucleic acid sequences have been identified and isolated, it is necessary to determine the sequence and analyze the properties. To do so, a number of molecular biological, biochemical and biotechnological standard methods are available to the expert.
The molecules that hybridize with the nucleic acid molecules, according to the invention, include also fragments, derivatives and allelic variants of the above-described DNA molecules that contain an ozone-responsive sequence in an active or inactivated form, or which are characterized by the fact that they no longer have such sequence. The term xe2x80x9cderivativexe2x80x9d means in this context that the sequences of these molecules distinguish themselves from the sequences of the above-described nucleic acid molecules in one or several positions and are to a great extent homologous with said sequences. Homology in this connection means a sequence identity of at least 40%, especially an identity of at least 60%, preferably above 80%, and especially preferable above 90%. The deviations from the above-described nucleic acid molecules can have been caused by deletion, addition, substitution, insertion or recombination.
With respect to the nucleic acid molecules which are homologous with the above-described molecules and which constitute derivatives of such molecules, it concerns usually variants of such molecules that constitute modifications which perform the same biological function. These may concern naturally occurring variations, e.g., sequences from other organisms, or mutations in which these modifications can have occurred naturally or were introduced through specific mutagenesis. Furthermore, the variations may concern synthetically produced sequences. With respect to the allelic variants, these may occur naturally as well as be synthetically produced variants, or variants produced by recombined DNA methods.
To prepare the insertion of foreign genes into higher plants or into the cells thereof, a large number of cloning vectors are available which contain a replication signal for E. coli and a marker gene to select transformed bacteria cells. Examples of such vectors are pBR322, pUC-series, M13mp-series, pACYC184, etc. The desired sequence can be inserted into the vector at a suitable restrictive cut. The plasmid obtained is used for the transformation of E. coli cells. Transformed E. coli cells are cultured in a suitable medium and subsequently harvested and lysed. The plasmid is recovered. Usually restrictive analyses, gel electrophoreses and other biochemical, molecular biological methods are applied as an analyzing method to characterize gained plasmid DNA. After each manipulation the plasmid DNA can be split and the gained DNA fragments can be linked with other DNA sequences. Each plasmid-DNA sequence can be cloned in identical or other plasmids.
Many well-known methods are available to introduce DNA into a plant host cell. The expert can determine, without difficulty, the respectively suitable method. These techniques include the following: the transformation of plant cells with T-DNA by using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation medium, the fusion of protoplasts, the direct gene transfer of isolated DNA in protoplasts, the electroporation of DNA, the introduction of DNA by means of the biolistic method, as well as other possibilities. In doing so, stable, as well as transient tranformants, can be produced.
When injecting and electroporating DNA into plant cells no specific demands per se is made on the plasmids used. The same applies to direct gene transfer. Simple plasmids as, e.g., pUC derivatives, can be used. If, however, whole plants are to be regenerated from cells transformed in this way, the presence of a selectable marker gene is required. The expert is familiar with the usual selection markers, and it does not pose a problem for him to select an appropriate marker. Selection markers in common use are those that make the transformed plant cells resistant to a biocide or an antibiotic, such as kanamycin, G418, bleomycin, hygromycin, methotrexate, glyphosate, streptomycin, sulfonyl-urea, gentamycin or phosphinotricin, etc.
Depending on the method selected for introducing genes into the plant cell, additional DNA sequences may be required. If, for example, the Ti or Ri-plasmid is used for the transformation of the plant cell, at least the right boundary, but frequently, however, the right and left boundary of the T-DNA, contained in the Ti and Ri-plasmid must, as flank region, be linked with the genes to be introduced.
If agrobacteria is used for the transformation, the DNA to be introduced must be cloned in special plasmids, i.e. either in an intermediary or in a binary vector. Due to sequences which are homologous with sequences in the T-DNA, the intermediary vectors can be integrated in the Ti- or Ri-plasmid of the agrobacteria through homologous recombination. In addition, the latter includes the vir-region required for the transfer of the T-DNA. Intermediary vectors cannot replicate in agrobacteria. The intermediary vector can be transferred to Agrobacterium tumefaciens (conjugation) by means of a helper plasmid. Binary vectors can replicate in E. coli as well as in agrobacteria. They contain a selection-marker gene and a linker or a polylinker which are framed in by the right and left T-DNA boundary region. They can be directly transformed into the agrobacteria (Holster et al (1978) Molecular and General Genetics 163, 181-187). The agrobacterium which serves as host cell shall contain a plasmid that has a vir-region. The vir-region is necessary for transferring the T-DNA to the plant cell. Additional T-DNA can be present. The agrobacterium, transformed in the manner described, is used for the transformation of plant cells.
The use of T-DNA for the transformation of plant cells has been thoroughly studied and is adequately described in EP 120 515; Hoekema in: The Binary Plant Vector System, Offsetdrokkerij Kanters B. V., Alblasserdam (1985) Chapter V; Fraley et al (1993) Crit. Rev. Plant. Sci., 4, 1-46 and An et al (1985) EMBO J. 4, 277-287.
For the transfer of the DNA to the plant cell, plant explantates can be appropriately cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes. Out of the infected plant material (leaf fragments, stem segments, roots, but also protoplasts or suspension-cultivated plant cells) whole plants can be regenerated in a suitable medium which can contain antibiotics or biocides for the selection of transformed cells. The regeneration of plants is carried out according to customary regeneration methods by using familiar culture media. Plants or plant cells obtained in the above-described manner can then be examined for the presence of the introduced DNA. Other possibilities for introducing foreign DNA by applying the biolistic method or through protoplast transformation are known (see, e.g., Willmitzer L. (1993) Transgenic Plants, in: Biotechnology, A Multi-Volume Comprehensive Treatise (H. J. Rehm, G. Reed, A. Pxc3xchler, P. Stadler, eds.) Vol. 2, 627-659, V. C. H. Weinheimxe2x80x94New Yorkxe2x80x94Baselxe2x80x94Cambridge).
Although the transformation of dicotyl plants or their cells via Ti-plasmid vector systems and with the help of Agrobacterium tumefaciens is well established, new studies indicate that also monocotyl plants or their cells are very receptive to transformation by means of Agrobacterium-based vectors (Chan et al (1993) Plant Mol. Biol. 22, 491-506; Hiei et al (1994) Plant J. 6, 271-282; Deng et al (1990) Science in China 33, 28-34; Wilmink et al (1992) Plant Cell Reports 11, 76-80; May et al (1995) Bio/Technology 13, 486-492; Conner and Domiss (1992) Int. J. Plant Sci. 153, 550-555; Ritchie et al (1993) Transgenic Res. 2, 252-265).
Alternative systems for transforming monocotyl plants or their cells are transformations by means of the biolistic setup (Wan and Lemaux (1994) Plant Physiol. 104, 37-48; Vasil et al (1993) (Bio/Technology 11, 1553-1558; Ritala et al (1994) Plant Mol. Biol. 24, 317-325; Spencer et al (1990) Theor. Appl. Genet. 79, 625-631), the protoplast transformation, the electroporation of partially permeabilized cells and the introduction of DNA by means of glass fibres.
The transformed cells grow within the plant in the usual manner (see also McCormick et al (1986) Plant Cell Reports 5, 81-84). The resulting plants can be cultivated normally and grafted with plants which have the same, transformed genetic trait or other genetic traits. The resulting hybrid individual plants have the respective phenotypical properties.
Two or more generations should be cultivated in order to ensure that the phenotypical characteristic is firmly maintained and propagated. Seeds should also be harvested in order to ensure that the respective phenotype or other characteristics are maintained.
By applying the usual methods, transgenic lines can be determined, which are homozygous for the new nucleic acid molecules and, furthermore, their phenotypical behaviour can be examined for an existing or non-existing ozone-response capacity and compared to that of hemizygous lines.
Naturally, plant cells which contain the nucleic acid molecules, according to the invention, can also be further cultivated as plant cells, (including protoplasts, calli, suspension cultures, etc.).
Another object of this invention is the use of the nucleic acid molecules, according to the invention, or fragments of such molecules, or the reverse complements of such molecules to identify and isolate homologous molecules which include ozone-responsive elements from plants or other organisms. As to the definition of the term xe2x80x9chomologyxe2x80x9d, please refer to the definition given earlier in the text.
The following examples serve the purpose of explaining the invention.